CN109971824A - A method of utilizing the mould quick detection bio-toxicity of fluorescein - Google Patents
A method of utilizing the mould quick detection bio-toxicity of fluorescein Download PDFInfo
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Abstract
The invention discloses a kind of methods using the mould quick detection bio-toxicity of fluorescein, are related to environmental monitoring technology field.The present invention includes step 1: mutational site is introduced into luc gene；Step 2: building cloning recombinant plasmids；Step 3: the PCR product of rite-directed mutagenesis is obtained；Step 4: building recombinant expression；Step 5: the genetic engineering bacterium of preparation expression luciferase；Step 6: luciferase and step 7: luciferase toxotest are extracted.Mutation luciferase luminous power prepared by the present invention can reach 100 times of wild-type luciferase, and have good thermal stability and resistance to acid and alkali；Meanwhile luciferase acute toxicity biological test method of the invention can the acute toxicity to light water pollutant directly tested, and more rapid sensitive has better application prospect in environmental protection and environmental monitoring field.
Description
Technical field
The invention belongs to environmental monitoring technology fields, utilize the mould quick detection bio-toxicity of fluorescein more particularly to a kind of
Method.
Background technique
It is more next to protect environment ecosystem health with to environment is organic and the gradually intensification of heavy metal pollution understanding
More toxotest methods are developed.Biological test be based on pollutant to microorganism growth and enzymatic activity inhibition come
Characterize the size of pollutant toxicity.Currently used parameter in bio kinetic model be using photobacteria method of testing, but it is thin due to shining
Bacterium is marine bacteria, when carrying out toxotest to fresh water, needs to adjust salinity, this is possible to change certain objects in fresh water
The dissociation and solubility of matter, to cause the deviation of test.Using enzymatic activity inhibition to toxicity test can not only from point
The sub horizontal upper toxicity for directly characterizing pollutant, and have the characteristics that quick and convenient reproducible.
Luciferase is one kind using ATP, fluorescein and oxygen as substrate, and magnesium ion is activator, glimmering in catalysis biological body
The general name of light element or the class of enzymes of fatty aldehyde oxyluminescence.Firefly luciferase is made of single polypeptide chain,
There is O2、Mg2+, ATP it is existing under the conditions of, form the adenylate fluorescein acylation compound with enzyme ining conjunction with substrate reactions, this is answered
Closing object becomes the oxyluciferin being active by oxidative decarboxylation, converts luminous energy for chemical energy, and release
Light quantum issues fluorescence.Different types of gene can be from a variety of light of firefly such as North America firefly, Japanese firefly, Beijing fireflies
It is extracted in worm.The relative molecular weight of the firefly luciferase extracted by firefly is about 60-64kD.
The activity of luciferase can be detected directly by the inhibition of bioluminescence, have high sensitivity, specificity
Many advantages, such as getting well, be swift in response, be easy to operate, directly the bio-toxicity of poisons in freshwater can be tested.But since worm is glimmering
Light element enzyme is more sensitive to temperature and pH value, it is therefore desirable to which thermal stability and the anti-external environment for improving luciferase are dry
The ability disturbed, and realize that it is used for the test of bio-toxicity.
Summary of the invention
In order to solve the above technical problems, the present invention is achieved by the following technical solutions:
The present invention is a kind of method using the mould quick detection bio-toxicity of fluorescein, comprising the following steps:
Step 1: mutational site is introduced into luc gene；Two groups of primers are designed according to luciferase encoding gene luc, are pressed
The sequence for obtaining the both ends luc is expanded according to overlap PCR program, splicing segment obtains mutated gene segment；
Step 2: building cloning recombinant plasmids；It is identified after mutated gene is connected with cloning vector, obtains clone's weight
Group plasmid；
Step 3: the PCR product of rite-directed mutagenesis is obtained；By the recombinant plasmid electricity of building go in DH5 α competent cell into
Row culture, by verifying, recycling to obtain PCR product after purification；
Step 4: building recombinant expression；The recombinant plasmid of PCR product after extraction purification, after digestion with expression
Carrier, which is connected, obtains recombinant expression；
Step 5: the genetic engineering bacterium of preparation expression luciferase；Recombinant expression electricity is gone into DH5 α competence
It is cultivated in cell, screening and identification goes out positive recombinant, and extracts recombinant plasmid and be transferred in F-strain BL21 (DE3),
Obtain can great expression luciferase genetic engineering bacterium；
Step 6: luciferase is extracted；Luciferase is extracted from the genetic engineering bacterium of expression luciferase；
Step 7: luciferase toxotest；Luciferase is added in test system, the big of luminous value is tested
It is small, compared with the control, the inhibiting rate that shines is calculated, so that EC50 value be calculated.
Further, the genetic fragment that the mutational site of the introducing is cloned is luc-I232K and luc-I232K-
F465R。
Further, cloning vector is pBBR1-MCS4 in the step 2.
Further, expression vector is pET-30a (+) in the step 4.
Further, test system includes 100 μm of ol/L of luciferin, bovine serum albumin(BSA) 0.05% in the step 7
(v/v), Triton X-100 0.01% (v/v), ATP 100 μm of ol/L, Mg2+7mmol/L and noxious material；The test body
System is 19:5 with luciferase volume ratio.
The invention has the following advantages:
Mutation luciferase luminous power prepared by the present invention can reach 100 times of wild-type luciferase, and have
There are good thermal stability and resistance to acid and alkali；Meanwhile it can based on this luciferase acute toxicity biological test method of the invention
It is directly tested with the acute toxicity to light water pollutant, and more rapid sensitive, in environmental protection, environmental monitoring field
There is good application prospect.
Certainly, it implements any of the products of the present invention and does not necessarily require achieving all the advantages described above at the same time.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, will be described below to embodiment required
Attached drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for ability
For the those of ordinary skill of domain, without creative efforts, it can also be obtained according to these attached drawings other attached
Figure.
Fig. 1 is PCR products electrophoresis map in comparative example one；
Fig. 2 is that bacterium colony PCR verifies electrophoretogram in comparative example one；
Fig. 3 is expression plasmid pBBR1-luc digestion verification electrophoretogram in comparative example one；
Fig. 4 is the luminous intensity comparison diagram of the luciferase of comparative example one and embodiment one and embodiment two；
Fig. 5 is the luciferase of comparative example one and embodiment one in 40 DEG C of tolerance comparison diagrams；
Fig. 6 be comparative example one from the luciferase of embodiment one the activity influence comparison diagram under different pH environment；
Fig. 7 is that the luciferase that the present invention is mutated responds the toxicity of heavy metal and organic matter.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts all other
Embodiment shall fall within the protection scope of the present invention.
Table 1. is used to construct band, and there are two the primer sequences of the luc gene in mutational site
Embodiment one
Preparation method containing a rite-directed mutagenesis luciferase encoding gene, comprising the following steps:
Step 1: mutational site is introduced into luc gene；The luc segment of Overlap PCR amplification acquisition rite-directed mutagenesis:
Design two groups of mutant primers, mutant primer is as shown in table 1, with first group of mutagenic primer luc-s1 and luc-s2 and luc-s3 and
luc-s4；It is expanded according to following condition: 95 DEG C of 5min of initial denaturation；94 DEG C of 0.5min are denaturalized, anneal 55 DEG C of 0.5min,
Extend 72 DEG C of 1min (30 circulations)；72 DEG C of ends extend 10min；
Primer luc-s1 and luc-s2 amplify S1S2, and primer luc-s3 and luc-s4 amplify S3S4, pcr amplification product
Through the recycling DNA kit recycling of Axygen company glue.
The above two PCR product S1S2 and S3S4 of recycling are mixed, the primer s1 and s4 that upstream and downstream is added carry out second
The PCR of wheel, PCR reaction are divided into twice: for the first time, 95 DEG C of 5min of initial denaturation；94 DEG C of 0.5min are denaturalized, are annealed 60 DEG C
0.5min extends 72 DEG C of 1.5min (3 circulations)；72 DEG C of ends extend 5min, and are not added and draw in first set reaction system
Object, Taq enzyme and pfu high fidelity enzyme.Second, add 95 DEG C of primer, Taq enzyme and pfu high fidelity enzyme initial denaturation 5min；Denaturation 94
DEG C 0.5min, anneal 55 DEG C of 0.5min, extends 72 DEG C of 1.5min (30 circulations)；72 DEG C of ends extend 10min.PCR amplification
Product is through the recycling DNA kit recycling of Axygen company glue；Obtain mutated gene luc-I232K；
Step 2: building cloning recombinant plasmids；Mutated gene luc-I232K and cloning vector pBBR1-MCS4 are used respectively
Hind III and Kpn I carries out 22 DEG C of double digestion overnight enzymes and connects, and obtains cloning recombinant plasmids；
Step 3: the PCR product of rite-directed mutagenesis is obtained；Cloning recombinant plasmids electricity is gone in DH5 α competent cell,
On the plate of the Amp containing antibiotic, 37 DEG C of selection cultures, picking monoclonal carries out bacterium colony PCR verifying and double digestion verifying, will verify
Correct positive strain is sequenced, and DH5 α-pBBR-luc-I232K is named as；With restriction enzyme Kpn I, Hind III into
As a result the identification of row double digestion, 1% agarose gel electrophoresis inspection generate 1568kb band, be consistent with expected size；
Step 4: building recombinant expression；Recombinant plasmid is extracted for positive strain DH5 α-pBBR-luc-I232K
Recombinant plasmid and pET-30a (+) are carried out double digestion with Hind III and Nde I, cut required purpose item by pBBR-luc-I232K
Band and carrier carry out glue recycling；It can refer to step listed by Genebase company plastic recovery kit；
Step 5: the genetic engineering bacterium of preparation expression luciferase；The recombinant expression that step 4 obtains is used again
22 DEG C of T4 ligase overnight enzymes connect, and enzyme-linked product electricity is gone in DH5 α competent cell, is selected on the plate of the Amp containing antibiotic
Culture is selected, utilizes the method screening and identification of PCR and double digestion to go out positive recombinant after picking monoclonal, and extract recombinant plasmid
PET-30a (+)-luc is transferred in F-strain BL21 (DE3), obtain can great expression luciferase gene work
Journey bacterium BL-pET-30a (+)-luc-I232K；
Step 6: luciferase is extracted；Above-mentioned thallus BL-pET-30a (+)-luc-I232K is centrifuged, in abandoning
Clearly, thallus is resuspended with lysis buffer, and ice-bath ultrasonic is broken, and centrifugation takes supernatant, sulfuric acid is added to connect precipitating, is centrifuged, is taken supernatant mistake
Ni-NTA affinity column washes column with the buffer containing imidazoles of 8 times of bed volumes, and the dense of imidazoles in eluent is then gradually increased
Degree is eluted, and is in charge of collection and is obtained saltant type luc-I232K luciferase；
Embodiment two
Preparation method containing two rite-directed mutagenesis luciferase encoding genes is as follows:
Using the pBBR-luc-I232K plasmid in embodiment one as template, as shown in table 1, with second group of mutagenic primer luc-
D1 and luc-d2 and luc-d3 and luc-d4 are expanded, 95 DEG C of 5min of initial denaturation；94 DEG C of 0.5min are denaturalized, are annealed 55 DEG C
0.5min extends 72 DEG C of 1min (30 circulations)；72 DEG C of ends extend 10min；Primer luc-d1 and luc-d2 expand d1d2,
Primer luc-d3 and luc-d4 amplify d3d4, and pcr amplification product is recycled through Axygen company glue recycling DNA kit.
The above two PCR product d1d2 and d3d4 of recycling are mixed, the primer d1 and d4 that upstream and downstream is added carry out second
The PCR of wheel, PCR reaction are divided into twice: for the first time, 95 DEG C of 5min of initial denaturation；94 DEG C of 0.5min are denaturalized, are annealed 60 DEG C
0.5min extends 72 DEG C of 1.5min (3 circulations)；72 DEG C of ends extend 5min, and are not added and draw in first set reaction system
Object, Taq enzyme and pfu high fidelity enzyme.Second, add primer, Taq enzyme and pfu high fidelity enzyme, 95 DEG C of 5min of initial denaturation；Denaturation 94
DEG C 0.5min, anneal 55 DEG C of 0.5min, extends 72 DEG C of 1.5min (30 circulations)；72 DEG C of ends extend 10min.PCR amplification
Product is through the recycling DNA kit recycling of Axygen company glue.It obtains containing mutational site I232K, the luciferase base of F465R
Cause.
The above-mentioned PCR product of recovery purifying carries out PCR product and plasmid pBBR1-MCS4 double with Hind III and Kpn I respectively
22 DEG C of digestion overnight enzymes connect, and electricity is gone in DH5 α competent cell, and on the plate of the Amp containing antibiotic, 37 DEG C of selection cultures are chosen
It takes monoclonal to carry out bacterium colony PCR verifying and double digestion verifying, correct positive strain will be verified and be sequenced；And it is named as
DH5α-pBBR-luc-I232K-F465R；
Double digestion identification is carried out with restriction enzyme Kpn I, Hind III, as a result 1% agarose gel electrophoresis inspection generates
1568bp band is consistent with expected size.
For positive strain DH5 α-pBBR-luc-I232K-F465R, recombinant plasmid pBBR-luc-I232K- is extracted
Recombinant plasmid and pET-30a (+) are carried out double digestion with Hind III and Nde I, cut required purpose band and carrier by F465R,
Glue recycling is carried out, can refer to step listed by Genebase company plastic recovery kit；Connected again with 22 DEG C of T4 ligase overnight enzymes, it will
Enzyme-linked product electricity is gone in DH5 α competent cell, in selecting culture on the plate of the Amp containing antibiotic, is utilized after picking monoclonal
The method screening and identification of PCR and double digestion goes out positive recombinant, and extracts recombinant plasmid pET-30a (+)-luc, is transferred to
To in F-strain BL21 (DE3), obtain can great expression luciferase genetic engineering bacterium BL-pET-30a (+)-luc-
I232K-F465R；
Above-mentioned thallus BL-pET-30a (+)-luc-I232K-F465R is centrifuged, supernatant, thallus cracking buffering are abandoned
Liquid is resuspended, and ice-bath ultrasonic is broken, and centrifugation takes supernatant, sulfuric acid is added to connect precipitating, is centrifuged, supernatant is taken to cross Ni-NTA affinity column, uses
The buffer containing imidazoles of 8 times of bed volumes washes column, and the concentration that imidazoles in eluent is then gradually increased is eluted, and is in charge of collection
Obtain saltant type luc-I232K-F465R luciferase.
The luciferase being prepared by embodiment one or embodiment two is added in toxotest system, is tested
System includes 100 μm of ol/L of luciferin, bovine serum albumin(BSA) 0.05% (v/v), Triton X-100 0.01% (v/v),
ATP 100μmol/L,Mg2+7mmol/L and noxious material, by poisonous substance (Cu2+, Cd2+, Cr6+, phenol, nitrobenzene) extremely required test
Concentration, 5 μ L luciferases are added in every 95 μ L system, the size of luminous value is tested after 15 seconds, compared with the control, calculate hair
Xanthophyll cycle rate, so that EC50 value is calculated, shown in chart 2；And the opposite hair of luciferase is tested under different concentration of poison
Luminous intensity, as shown in Figure 7.
EC50 value of 2. luciferase of table to different heavy metals and organic matter
Comparative example one
The preparation method of unmutated coding luciferase bacterial strain is as follows:
Specific primer luc-KpnI, luc-Hind III is designed, luciferase is encoded by the method massive amplification of PCR
Luc gene.Using pGEM-luc as template, PCR reaction condition: 95 DEG C of 5min of initial denaturation；It is denaturalized 94 DEG C of 0.5min, annealing 55
DEG C 0.5min extends 72 DEG C of 1min (30 circulations)；72 DEG C of ends extend 10min.PCR product carries out 1% Ago-Gel
Electrophoresis confirms PCR product size.
PCR product and plasmid pBBR1-MCS4 are carried out 22 DEG C of double digestion overnight enzymes with Hind III and Kpn I respectively to connect, electricity
It goes in DH5 α competent cell, on the plate of the Amp containing antibiotic, 37 DEG C of constant temperature selection cultures, picking monoclonal carries out bacterium
PCR verifying and double digestion verifying are fallen, correct positive strain will be verified and be sequenced.
For positive strain DH5 α/pBBR-luc, recombinant plasmid pBBR-luc is extracted, by recombinant plasmid and pET-30a (+)
Double digestion is carried out with Hind III and Nde I, cuts required purpose band and carrier, carries out glue recycling, referring to Genebase company glue
Step listed by QIAquick Gel Extraction Kit；Connected again with 22 DEG C of T4 ligase overnight enzymes, enzyme-linked product electricity gone in DH5 α competent cell,
In selecting culture on the plate of the Amp containing antibiotic, gone out after picking monoclonal using the method screening and identification of PCR and double digestion positive
Recon, and recombinant plasmid pET-30a (+)-luc is extracted, it is transferred in F-strain BL21 (DE3), obtaining can be big
The genetic engineering bacterium BL-luc of amount expression luciferase；
Genetic engineering bacterium BL-luc thallus is centrifuged, supernatant is abandoned, thallus is resuspended with lysis buffer, and ice-bath ultrasonic is broken,
Centrifugation, takes supernatant, sulfuric acid is added to connect precipitating, centrifugation takes supernatant to cross Ni-NTA affinity column, with 8 times of bed volumes containing imidazoles
Buffer washes column, and the concentration that imidazoles in eluent is then gradually increased is eluted, and is in charge of collection, obtains unmutated worm fluorescence
Plain enzyme, i.e. wild type luciferase.
It using pGEM-luc as template, is expanded with the specific primer of luc gene, amplified production is solidifying through 1% agarose
Gel electrophoresis analysis, it is seen that the DNA fragmentation of a treaty 1.6kb, it is consistent with luc gene size, as shown in Figure 1；By PCR product luc
Sunk with Kpn I and III double digestion of Hind again after, it is connected with the carrier through identical digestion, recombinant plasmid pBBR-luc is obtained, turns
Enter Escherichia coli, after bacterium colony PCR is verified and double digestion is verified, display target gene successful clone to carrier pBBR1-MCS4
In, as shown in Figures 2 and 3；It send positive bacteria bacterium solution to be sequenced, the sequence announced in sequencing result and NCBI is compared, it is homologous
Property is up to 100%.
High efficient expression is carried out with similarity condition by the way that engineering strain will be obtained in embodiment one, embodiment two and comparative example one
It is compared with after extraction purification, steps are as follows；
Under conditions of 37 DEG C and 240rpm, strain is accessed in the LB culture solution containing kanamycins respectively, is incubated overnight,
Then it accesses in the fresh LB culture solution containing kanamycins in the ratio of 1:100, is cultivated under the conditions of 37 DEG C and 240rpm；Logarithm
When growth medium OD=0.3-0.4,1.0mM IPTG induction is added, is cultivated under the conditions of 28 DEG C and 240rpm, harvests thallus, carried out
The extraction purification of luciferase.
Luciferase gene is connected on the carrier pET-30a (+) of high efficient expression in the present embodiment, luc gene
It is no longer influenced by the regulation of other natural promoters, and the only regulation by the strong promoter T7 on plasmid, luc gene have obtained greatly
The expression of amount, and luciferase shows high activity.Since the carrier pET-30a (+) that the present invention uses is low-copy matter
Grain, can mitigate the burden of strain growth, thus the growth of transgenic strain with compare bacterium and there is no conspicuousness poor early period growing
Not, and expressed luciferase is present in intracellular with soluble state, is readily available after microorganism collection and breaking-wall cell
The zymoprotein of high concentration, and improve the yield of enzyme；Meanwhile albumen expressed by pET-30a (+) contains histidine tag, it can be with
It is quickly purified with Ni affinity chromatography；
Respectively by the bacterial strain of above-mentioned high efficient expression, supernatant is abandoned in centrifugation；Thallus is centrifuged again after being resuspended with PBS, abandons supernatant,
Somatic cells are resuspended in 1 × Binding Buffer of ice bath, serpin (PMSF) is added in ice bath
Ultrasonication；The centrifugation of bacteria breaking liquid；It collects supernatant to be purified using nickel affinity chromatography system, enzyme solution after purification is added to
In luminescence reagent, luminous intensity is detected to examine the activity of luciferase.It is as follows specifically to cross ni-sepharose purification method:
(1) it is gently mixed by inversion solidification Ni2+ resin, 2mL is taken to be packed into chromatographic column, resin natural subsidence；
(2) with the aseptic water washing resin of 3 times of column volumes；Volume after resin sedimentation is 1 column volume；
(3) resin is balanced with the Binging Buffer (pH7.8) of 3 times of column volumes；
(4) the Binging Buffer on resin drains into column top；
(5) by sample upper prop, flow velocity is adjusted to 10 column volumes per hour；
(6) column is washed with the Binging Buffer (pH7.8, imidazole concentration 10mmol/L) of 6 column volumes；
(7) column is washed with the Washing I (imidazole concentration 20mmol/L) of 6 column volumes, connects 6 pipe filter liquors (1mL/ pipe)；
(8) column is washed with the Washing II (imidazole concentration 50mmol/L) of 6 column volumes, meets 6 pipe filter liquor (1mL/
Pipe)；
(9) column is washed with the Elut I (imidazole concentration 100mmol/L) of 6 column volumes, connects 6 pipe filter liquors (1mL/ pipe)；
(10) column is washed with the Washing I (imidazole concentration 250mmol/L) of 6 column volumes, meets 6 pipe filter liquor (1mL/
Pipe)；
(11) 1,3, the 5 pipe filter liquors for being connect (7)-(10) carry out the detection of fluorescent value.
The luciferase that purification is obtained, is added in luminescence reagent, characterizes worm fluorescence as index using luminous intensity
The activity of plain enzyme；The luciferin enzyme solution for the mutation for respectively taking purification to obtain and unmutated wild type luciferin enzyme solution are added
Into luminescence reagent, the variation of luminous intensity is detected；
As shown in figure 4, the luminous value of wild type only has 10000, and the luminous intensity of saltant type luc-I232K-F465R reaches
It is 80 times of wild type to 800000, the luminous value of saltant type luc-I232K has been even up to 1000000, is wild type
100 times；Prove that saltant type luciferase prepared by the present invention has stronger luminous power；
Heat stability test: the luciferin enzyme solution for the mutation that extraction purification takes purification to obtain and unmutated wild type worm
Fluorescein enzyme solution, is added in luminescence reagent, is incubated for 5min respectively under 40 DEG C of environment, takes out after 10min, 20min, detection hair
Luminous intensity.
As shown in figure 5, unmutated wild type luciferase enzyme activity under 40 DEG C of heat-retaining conditions reduces rapidly, at 10 points
Zhong Hou, fluorescent value only has 100, and the reduction of the enzyme activity of two kinds of mutant luciferases prepared by the present invention is relatively slow, and 10 points
Luminous intensity after clock is 20 times of wild type there are also 20000；Illustrate the saltant type luciferin enzyme solution prepared through the invention
With stronger thermal stability.
Acid and alkali-resistance detection: the luciferin enzyme solution and unmutated wild type luciferin enzyme solution of mutation are respectively taken, is added to
In luminescence reagent, pH is adjusted respectively to 6.0,7.0,7.8,9.0, with the NaOH solution of the HCl solution of 1mol/L and 1mol/L
In 4 DEG C of incubation 30min, with fluorescence radiation detector test fluorescence intensity；
As shown in fig. 6, the fluorescent value of enzymatic reaction of the unmutated wild type luciferase in pH6.0-9.0 exists
Between 1000-20000, and the fluorescent value for passing through the enzymatic reaction in pH6.0-9.0 of the luciferase of rite-directed mutagenesis exists
100000-1000000, illustrate the saltant type luciferin enzyme solution prepared through the invention has stronger adaptability to pH.
In the description of this specification, the description of reference term " one embodiment ", " example ", " specific example " etc. means
Particular features, structures, materials, or characteristics described in conjunction with this embodiment or example are contained at least one implementation of the invention
In example or example.In the present specification, schematic expression of the above terms may not refer to the same embodiment or example.
Moreover, particular features, structures, materials, or characteristics described can be in any one or more of the embodiments or examples to close
Suitable mode combines.
Present invention disclosed above preferred embodiment is only intended to help to illustrate the present invention.There is no detailed for preferred embodiment
All details are described, are not limited the invention to the specific embodiments described.Obviously, according to the content of this specification,
It can make many modifications and variations.These embodiments are chosen and specifically described to this specification, is in order to better explain the present invention
Principle and practical application, so that skilled artisan be enable to better understand and utilize the present invention.The present invention is only
It is limited by claims and its full scope and equivalent.
Claims (5)
1. a kind of method using the mould quick detection bio-toxicity of fluorescein, which comprises the following steps:
Step 1: mutational site is introduced into luc gene；Two groups of primers are designed according to luciferase encoding gene luc, according to
The amplification of overlap PCR program obtains the sequence at the both ends luc, and splicing segment obtains mutated gene segment；
Step 2: building cloning recombinant plasmids；It is identified after mutated gene is connected with cloning vector, obtains clone's recombination matter
Grain；
Step 3: the PCR product of rite-directed mutagenesis is obtained；The recombinant plasmid electricity of building is gone in DH5 α competent cell and is trained
It supports, by verifying, recycling to obtain PCR product after purification；
Step 4: building recombinant expression；The recombinant plasmid of PCR product after extraction purification, after digestion with expression vector
It is connected and obtains recombinant expression；
Step 5: the genetic engineering bacterium of preparation expression luciferase；Recombinant expression electricity is gone into DH5 α competent cell
In cultivated, screening and identification goes out positive recombinant, and extracts recombinant plasmid and be transferred in F-strain BL21 (DE3), obtains
Can great expression luciferase genetic engineering bacterium；
Step 6: luciferase is extracted；Luciferase is extracted from the genetic engineering bacterium of expression luciferase；
Step 7: luciferase toxotest；Luciferase is added in test system, the size of luminous value is tested,
Compared with the control, the inhibiting rate that shines is calculated, so that EC50 value be calculated.
2. a kind of method using the mould quick detection bio-toxicity of fluorescein according to claim 1, which is characterized in that institute
Stating the genetic fragment that the mutational site of introducing is cloned is luc-I232K and luc-I232K-F465R.
3. a kind of method using the mould quick detection bio-toxicity of fluorescein according to claim 1, which is characterized in that institute
Stating cloning vector in step 2 is pBBR1-MCS4.
4. a kind of method using the mould quick detection bio-toxicity of fluorescein according to claim 1, which is characterized in that institute
Stating expression vector in step 4 is pET-30a (+).
5. a kind of method using the mould quick detection bio-toxicity of fluorescein according to claim 1, which is characterized in that institute
Stating test system in step 7 includes 100 μm of ol/L of luciferin, bovine serum albumin(BSA) 0.05% (v/v), Triton X-
1000.01% (v/v), ATP 100 μm of ol/L, Mg2+7mmol/L and noxious material；The test system and luciferase body
Product is than being 19:5.
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| US20100068739A1 (en) * | 2005-08-10 | 2010-03-18 | Laurence Tisi | PH Tolerant Luciferase |
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| US20100068739A1 (en) * | 2005-08-10 | 2010-03-18 | Laurence Tisi | PH Tolerant Luciferase |
| US20110081298A1 (en) * | 2009-10-01 | 2011-04-07 | Andrew Tsourkas | Ratiometric bioluminescent sensor for imagining oxidative stress |
| CN105807064A (en) * | 2014-12-31 | 2016-07-27 | 中国科学院深圳先进技术研究院 | Luciferase complementary quantum dot biosensor as well as construction method and application thereof |
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Application publication date: 20190705 |